1. Field of the Invention
The present invention relates to the biotechnology field, more specifically to plant genetic engineering. The invention provides useful DNA sequences and constructs to obtain transgenic plants with a shorter life cycle and more tolerance to herbicides. More specifically, the invention provides new constructs that contain the sunflower Hahb-10 gene coding sequence, and a method to obtain plants with high tolerance to herbicides that produce oxidative stress, such as methyl viologen, and with a reduced life cycle in comparison to wild-type plants of the same species using these constructs.
2. Background Art
Homeobox Genes and their Participation in Plant Development
Plant development is determined by spatial and temporal gene expression programs. These programs are governed by regulatory proteins that act as transcription factors.
One distinctive characteristic of plants is that they can alter their development in response to environmental conditions in order to adapt to them. The first link in this response is a chain reaction triggered by a transcription factor that shoots biochemical and physiological changes in the plant. The knowledge of the transcription factors that drive these responses would be useful for the production of transgenic plants capable of adapting to unfavorable environmental conditions.
Transcription factors have specific DNA binding domains for particular sequences in the DNA promoter region in their target genes. The homeodomain is a 60-amino acid protein motif present in lots of eukaryotic transcription factors which are generally involved in the regulation of developmental processes. (Gehring, 1987). Homeodomains are present in almost every eukaryotic organism that has been investigated. Several homeobox genes have been isolated from fungi, animals and plants (Gehring et al., 1994).
In plants, several families of homeodomain proteins have been described (Chan et al., 1998). One of these families, named HD-Zip, comprises proteins with a typical leucine zipper motif adjacent to the C-terminal end of the homeodomain. As expected, these proteins bind DNA as dimers. The removal of the leucine zipper or the introduction of extra amino acids between the homeodomain and the leucine zipper significantly reduces binding affinity, indicating that the leucine zipper is responsible for the correct positioning of the homeodomain for efficient binding. The analysis of binding at different protein concentrations suggests that dimer formation is a prerequisite for DNA binding (Sessa et al., 1993; Palena et al., 1999).
It has been suggested, and subsequently supported by experimental evidence, that HD-Zip proteins are involved in regulating developmental processes associated with the response of plants to environmental conditions (Carabelli et al., 1993; Chan et al., 1998, Deng et al., 2002; Hanson et al., 2001; Himmelbach et al., 2002; Sawa et al., 2002; Schena et al., 1993; Soderman et al., 1999).
The expression of a member of Arabidopsis subfamily II, Athb-2/HAT4, is regulated by far-red light and its function is related to shade avoidance responses (Carabelli et al., 1993; Morelli and Ruberti, 2000; Morelli and Ruberti, 2002; Steindler et al., 1999). HAT2 is also a member of Arabidopsis subfamily II, and it has been characterized as an auxin inducible gene by DNA microarray screening (Sawa et al., 2002).
The isolation and characterization of several homeobox genes from sunflower has been previously reported (Chan and Gonzalez, 1994; Gonzalez and Chan, 1993; Gonzalez et al., 1997; Valle et al., 1997). Hahb-4 is a member of subfamily I and its function is related to water stress response (Dezar et al., 2005a; Dezar et al., 2005b; Gago et al., 2002).
Life Cycle of Plants
A plant's life cycle begins with seed germination and continues with seedling growth, until it constitutes a mature plant that flowers and produce fruits which contain seeds that will begin the plant cycle once again. Obtaining plants with shorter life cycles would be of major importance in modern agriculture.
Weeds and Herbicides
Weeds are unwanted plants that reduce both crop yield and quality by competing with the crop for water, nutrients and light. Weeds shade crops, take their water and nutrients, and make harvesting difficult.
It is well known that weeds might compete with the crop for nutrients, cause toxicity to livestock, might act as host for many different insects and plagues, interfere with the harvest task, and release several substances that could affect the normal development of crops of interest. This is why, for fanning activity, the weeds are seen as harmful and unwanted plants that should be eliminated from the countryside.
Several chemical substances, called herbicides, have been employed in the control of such weeds, causing their death or inhibiting their growth, without affecting the crop of interest. Paraquat is an herbicide in which the active ingredient is methyl viologen. This herbicide protects crops by controlling a wide range of annual and certain perennial weeds. Extensive use of Paraquat has resulted in weed resistance. In those cases where resistant biotypes have evolved, they have had no significant agricultural impact.
The active ingredient of Paraquat is methyl viologen, a redox cycler that produces specific reactive oxygen species, superoxide, in treated cells, generating a strong oxidative burst in sensitive plants. Superoxide generates cellular membrane weakening with subsequent ion imbalance, and the bleaching phenomenon in the sheets by the destruction of chlorophyll molecules.
The choice of treatment with herbicides varies according to the crop being cultivated. The quantities to use depend on the development stage of the plants and on the weed to be eliminated. Great efforts have been dedicated to the obtaining of species with good tolerance to herbicide treatment in order to apply those herbicides without damaging the crop of interest.
Different methodologies of vegetal improvement have contributed in this sense. However, we do not count the genotypes of all cultivable species that present resistance, given that neither classic cross-linking methods, nor molecular markers attend selection, or obtaining of mutants has been successful. Genetic engineering has contributed, in this sense, incorporating unique genes in the plants of interest generating resistance to the treatment with those compounds capable of eliminating weeds.
One of the main objectives of agro biotechnology is the identification of genes that generate plant resistance or tolerance to different herbicides to allow their use without detrimental effects on crops of interest.